NMR characterization of three forms of ferredoxin from Desulphovibrio gigas, a sulphate reducer

A NMR and magnetic susceptibility study of the oxidized and reduced states of three different oligomers (forms) of a [4Fe-4S] ferredoxin protein from Desulphovibrio gigas, FdI, FdI′, and FdII was carried out. FdI and FdI′ are different trimers and FdII a tetramer of the same basic subunit. A probable assignment of the contact shifted resonances is indicated. Since the temperature dependences of the contact shifted resonances associated with each [4Fe-4S] are not all similar a delocalized model for the spin densities on the 4Fe does not apply. The exchange rate between oxidized and reduced states is slow on the NMR time scale. The three oligomers are not magnetically equivalent. Using the “three state hypothesis” terminology it is shown that FdI_ox is predominantly in the C^2? state and changes upon reduction into the C^3? state, while FdII_ox is in the C^? state and changes into the C^2? state. FdI′ does not easily fit into this classification. This study shows a similarity of magnetic behaviour between FdI and bacterial ferredoxins (e.g. Bacillus polymyxa) and between FdII and HiPIP from Chromatium sp.. The influence of the quaternary structure on the stabilization of the different oxidation states of ferredoxins as well as on their redox potentials is discussed.     

Heterogeneity of urinary oligosaccharides from mannosidosis: Mass spectrometric analysis of permethylated Man9, Man8, and Man7 derivatives

Three oligosaccharides isolated from the urine of a patient suffering from mannosidosis, a Man₉GlcNAc, a Man₈GlcNAc, and a Man₇GlcNAc, were analyzed by electron impact mass spectrometry at 20 eV after reduction with NaB²H₄ and permethylation. Molecular ions were observed at $\text{m}\text{e}$ 2144, 1940, and 1736, respectively. In the high-mass range very intense ions were found at M-45. The mass spectrum of the homogenous decasaccharide Man₉GlcNAc_1D contains ions that could be attributed to specific parts of each of the three antennae of the molecule. Thus characteristic key ions were recognized. With the aid of these key ions the spectra of the nona- and octasaccharide mixtures could be evaluated in a qualitative and a semiquantitative way. In the nonasaccharide all three possible isomers that can be produced by cleavage of one of the three terminal α-mannoses are present, although in differing amounts. However, only five of the six possible isomers of the octasaccharide could be detected.     

Fusion of sphingomyelin vesicles induced by proteins from Taiwan cobra (Naja naja atra) venom. Interactions of zwitterionic phospholipids with cardiotoxin analogues

Egg sphingomyelin vesicles were used to assay aggregation/fusion activities of proteins from Taiwan (Naja naja atra) venom to avoid the problem of phospholipase A2 contamination during protein purification. It led to the identification of a new cardiotoxin (CTX) analogue protein (CTX V) with major aggregation/fusion, but few hemolysis, activities. On the contrary, cardiotoxin (CTX III) induced significant hemolysis of human red blood cells but exhibited few aggregation/fusion activities. To study the structure/activity relationship of these CTX-induced processes, the amino acid sequence of CTX V was determined and its aggregation/fusion activity was compared with that of CTX III by transmission electron microscopy, quasielastic laser light scattering, differential scanning calorimetry, and fluorescence spectroscopy. The results show that the CTX-induced fusion process at temperatures slightly above that of the gel to liquid-crystalline phase transition of sphingomyelin vesicles can ultimately convert small sonicated vesicles into large fused vesicles with sizes of 1-2 microns. The abilities of CTX V to induce the leakage of sphingomyelin vesicles content and to cause the fusion of vesicles are approximately 10-fold higher than those of CTX III. Based on the CTX structures determined in the present and other studies, it is suggested that the amino acid residue X within the well conserved sequence of -Cys-Pro-X-Gly-Lys-Gln-Leu-Cys- plays a role in the interaction of CTX with lipid molecules. The lipid phase transition could further enhance the protein-lipid interaction in the process leading to the fusion of vesicles.     

Synergistic Regulation of Cytosolic Ca2+ Concentration in Mouse Astrocytes by NK1 Tachykinin and Adenosine Agonists

The effects on cytosolic Ca2+ concentration of 2-chloroadenosine and [L-Pro9]-substance P, a selective agonist of NK1 receptors, were investigated on astrocytes from embryonic mice in primary culture. Cells responded to [L-Pro9]-substance P with a transitory increase in cytosolic Ca2+ which was of shorter duration when external Ca2+ was removed. A transient response to 2-chloroadenosine alone occurred. When simultaneously applied, [L-Pro9]-substance P and 2-chloroadenosine evoked a prolonged elevation of cytosolic Ca2+ (up to 30 min). This phenomenon was dependent on the presence of extracellular Ca2+, but insensitive to dihydropyridines, La3+, and Co2+, excluding the implication of voltage-operated Ca2+ channels. Arachidonic acid also induced a sustained elevation of cytosolic Ca2+, but did not increase further the response evoked by [L-Pro9]-substance P and 2-chloroadenosine. The activation of protein kinase C by a diacylglycerol analogue mimicked the effect of [L-Pro9]-substance P in potentiating the 2-chloroadenosine-evoked response. Like 2-chloroadenosine, pinacidil, which hyperpolarizes the cells by opening K+ channels, prolonged the elevation of cytosolic Ca2+ concentration induced by [L-Pro9]-substance P. Conversely, depolarization with 50 mM KCl canceled the effects of either pinacidil or 2-chloroadenosine applied with [L-Pro9]-substance P. Pertussis toxin pretreatment suppressed all the effects induced by 2-chloroadenosine.     

Experimental silver bioaccumulation in the polychaetePomatoceros triqueter (L.)

Summary The tubicolous polychaetePomatoceros triqueter was exposed for 6–7 weeks to 200 or 400 g · l^–1 silver introduced as the nitrate into sea water. Survival conditions and mortality were evaluated and silver bioaccumulation analysed by atomic absorption spectrometry. Characteristic morphological lesions were recognized. Histopathologic examination was performed on paraffin or semi-thin sections and at the ultrastructural level. Histochemical examination mainly concerned the metals, reducing groups and sulfur-containing proteins. Microanalytical study involved the use of a wavelength-dispersive X-ray spectrometry microprobe and ion microanalyzer, and the use of an energy-dispersive X-ray spectrometry microprobe at the ultrastructural level. Our results emphasize the role of the branchial crown for metal penetration. Its cuticle accumulates silver as a metal, in particulate form. The internal accumulation of mainly extracellular deposits concerns the basement membranes and connective tissue present in the axis of the branchial crown filaments, or surrounding the nephridial pouches and the gut sinus. The carrier role of the closed vascular system is suggested by ultrastructural observations. The silver route from transepithelial uptake to nephridial excretion involves at least two intracellular transits, plus the vascular mesothelium. Nephridia play a role in silver storage (lysosomes) and elimination (concretions). In all parts internal to the crown cuticle, silver is at least partly associated with protein SH-groups (metallothionein-like); deposits can be enriched with silver sulfide and metallic silver.     

Design, synthesis and cytotoxic evaluation of keto-C-glycoside fatty acid conjugates.

Keto C-glycoside-fatty acid conjugates were synthesized from 6-hydroxy 2- and 4-keto unsaturated D-C-glycosides. These compounds were tested for cytotoxic activity against LFCl₂A cells (Rat hepatocarcinoma cells). The introduction of a lipid chain to 2-keto C-glycosides induced a drop in the cyctotoxic activity of these compounds. On the other hand 4-keto unsaturated C-glycoside-fatty acid conjugates possessed IC₅₀ values of 0.7–0.001 μM with 21 being the most potent.     

Surface heterogeneity of rat sperm during maturation

Rat sperm isolated from the caput and caudal epididymis and the vas deferens were subjected to multiple partition in aqueous two-phase systems. The technique was used to reveal heterogeneity of a sperm population with respect to particular surface properties. Sperm from all three regions gave broad distributions indicative of heterogeneous cell populations. Greatest heterogeneity was observed for cauda sperm with caput and was sperm producing similar distributions.Following multiple partition sperm from different regions of the distribution profiles were immunostained with three antibodies known to recognise maturation antigens. The results show that some antigens are acquired during epididymal transit whilst others are present throughout.The partition (surface heterogeneity) seen cannot therefore be explained solely by the distribution of the antigens recognised by 2D6, 6B2 and 3D5.